Highly attenuated rubella virus vaccine and production thereof

ABSTRACT

A NOVEL, HIGHLY ATTENUATED LIVE RUBELLA VACCINE IS PREPARED BY SUBJECTING A SPECIFIC RUBELLA VIRUS STRAIN TO AT LEAST 10 PASSAGES OF CULTIVATION IN TISSUE CONTAINING LIVING CELLS OF A WARM BLOODED ANIMAL AT 28*C. TO 36*C. UNTIL THE DESIRED DEGREE OF ATTENUATION IS ATTAINED.

United States Patent 3,655,872 HIGHLY ATIENUATED RUBELLA VIRUS VACCINEAND PRODUCTION THEREOF Reisaku Kono, Tokyo, Shigeo Yarnamoto, Yamaguchi,and Hideaki Yaoi, Nara, Japan, assignors to Takeda Chemical Industries,Ltd., Osaka, Japan No Drawing. Filed Apr. 1, 1969, Ser. No. 812,385

Int. Cl. C12k 5/00 US. Cl. 424-89 2 Claims ABSTRACT OF THE DISCLOSURE Anovel, highly attenuated live rubella vaccine is prepared by subjectinga specific rubella virus strain to at least passages of cultivation intissue containing living cells of a warm blooded animal at 28 C. to 36C. until the desired degree of attenuation is attained.

This invention relates to a highly attenuated live rubella vaccine whichis not only novel and effective but also further attenuated, i.e. farless in side-effects, than those which have been hitherto on trial.

Rubella is a measles-like infectious disease caused by the infection ofrubella virus; that is characterized by slight fever, skin eruption andlymphadenopathy which appear after an incubation period of a few weeks.Wh le rendering no serious symptoms in case of infants and ch11- dren,the disease tends to be severer in adults with accompaniment ofarthritis, arthralgia or the like. Particularly when pregnants sufferfrom this disease at an early stage of the pregnancy, the infectionextends beyond the placenta to fetus, resulting baby with the congenitalrubella syndrome, such manifestations as cataracts, congenital heartfailure and hearing difliculty, at high rate. Moreover, there isobserved a tendency that extensive outbreaks of rubella occurperiodically. Therefore, the development of an effective control measureof rubella has been a desideratum among physicians.

It goes without saying that the rubella is desirably to be controlled byany means, and a possibility of rubella vaccination has been extensivelyinvestigated after the discovery of rubella virus by Weller et a1. andParkman et al. in 1962. As the results, it has hitherto been succeededin allowing a rubella virus to propagate in a mammalian or fowlembryonic tissue culture, and it has also been shown that the rubellavirus can be attenuated through serial passages in said culture to agrade of virulence which permits the inoculation to humans as a liveattenuated vaccine and renders the immunity to the vaccines withoutserious side effects.

It has been clinically approved that when an attentuated live rubellavaccine is inoculated into human infants and children, the correspondingantibody response can be elicited without giving untoward clinicalreactions, and also that those who have acquired the antibody can beprotected against suffering from rubella. The rubella virus isoccasionally recovered from the throat of the vaccinees in laboratories,but it is known that the contact infection has never been found insusceptible children and adults living near by, since the contacts havenever shown clinical attack of German measles and serological evidenceof rubella virus infection.

On the other hand, however, when the hitherto-known attenuated li-verubella virus vaccines are inoculated into adults, particularly intoWomen of childbearing age, the reactions are different from those ofinfants and children, and there are observed frequently so-calledrubella-like symptoms such as fever, malaise, skin eruption, arthritis,arthralgia and the like. Therefore, there still remains a difficultproblem in inoculating the known attenuated live rubella virus vaccinesinto susceptible adults.

Moreover, it is not known whether the known vaccine is completely devoidof teratogenicity or fetal toxicity for the offspring, when it isinoculated into the pregnants.

During the present inventors comparative investigations on biologicalbehaviors of many rubella virus strains of various passage histories,some being isolated by them and others being contributed by otherresearchers in the world, they have found that some congenitalabnormalities similar to the congenital rubella syndrome observed inhumans are produced in the offspring by inoculating the doe with arubella virus at an early stage of its pregnancy.

The present inventors further noted that among those rubella virusestested, rubella virus, Strain To3-36, one of those which had beenisolated by one of the present inventors, shows substantially no fetaltoxicity not only in rabbits but mice and rats (refer to Tests 1 and 2detailed hereinafter). Proceeding attentuation of this strain, theyfinally succeeded in producing a highly attenuated rubella virus vaccinewhich does not give such undesirable side effects against human adultsas mentioned above.

The principal object of the present invention is therefore to provide ahighly attenuated live rubella virus vaccine which is novel andeffective but does not produce, after vaccinated, the rubella-likesymptoms and the teratogenicity or the fetal toxicity, both having beendifficult to be completely eliminated from the hitherto known rubellavirus vaccines.

Another object of this invention is to provide a method for theproduction of said novel and elfective, highly attenuated live rubellavirus vaccine with ease and at an economically realizable cost.

Further object of the present invention is to provide an effective andsafer immunological measure for preventing human adults, particularlywomen of childbearing age, from rubella virus infection without theteratogenicity or the fetal toxicity and untoward reactions.

Said objects are realized by subjecting rubella virus Strain To-336passaged at least 10 times through per se known means to furtherpassages of cultivation in a tissue culture prepared from awarm-bloooded animal at a temperature of about 2 8 C. to about 36 C.,until sufliciently attenuated.

The rubella virus Strain To-336 has been isolated by one of the presentinventors from a throat swab of a girl suflering from German measles inJapan and its subculture has been deposited at American Type CultureCollection, Maryland, U.S.A. under the accession number of ATCC-VR-553.

For example, Strain To-336 is inoculated in a tissue culture containingsuitable primary cells of a warmblooded animal, and is incubatedstationarily or rotationally at a temperature between about 28 C. andabout 36 C. (usually about 29 C. to about 32 C.) for about to about days(usually about one week), and the propagated virus is then transferredinto a fresh tissue culture containing such primary cells as mentionedabove for the subsequent passage of cultivation. As such passage isrepeated successively, the attenuation proceeds rapidly. The cells forthe tissue culture are suitably chosen among tissues of cells ofwarm-blooded animals, i.e. mammals and fowls, and are exemplified byprimary kidney cells of various animals (such as monkeys, bovines, pigs,rabbits and dogs), embryo cells of various poultries (such as chickensor ducks) and the like.

Also, for carrying out the serial passages of cultivation, there may beemployed human fetal diploid cell cultures.

Alternatively, Strain To-336 is inoculated in the amniotic cavity of anembryonated egg (e.g. on the 7th or 8th day of hens egg or ducks egg)and incubated at a temperature of from about 30 C. to about 36 C. forabout 7 to about 10 days. The inoculated amnion is then removedaseptically, and triturated in a suitable medium such as Hanks balancedsalt solution (detailed below) or the like to prepare about 10%suspension, followed by centrifugation to separate the supernatantfluid. The supernatant fluid contains propagated virus, and can besubjected to the subsequent passage of cultivation in the same manner asmentioned above, whereby the attenuation rapidly proceeds.

For the new passage of cultivation with a tissue culture or anembryonated egg, the culture fluid of the preceding passage is usuallydiluted about 10 times by volume and the diluted fluid is employed asthe seed virus to be inoculated. However, if desired, use may be made ofa non-diluted culture fluid, or the so-called limiting dilution methodmay be occasionally applied. The passage or passages with amnioticcavity may be subsequently followed by the passage or passages with atissue culture containing the primary cells of a warm-blooded animal orthe human diploid cells, and vice versa.

In the culture medium for conducting a passage of cultivation, there maybe added an antibiotic or antibiotics such as streptomycin,dihydrostreptomycin, neomycin or penicillins so that the culture may beprevented from the propagation of adventitious microorganisms by anaccidental contamination.

In such a manner, rubella virus Strain To-336 is repeatedly subjected tothe passages of cultivation until it is confirmed that the virus hassufliciently been attenuated, by a tentative inoculation intoseronegative human body susceptible to rubella virus.

Thus attenuated Strain To-336 is used as the seed virus for theproduction of the highly attenuated rubella virus vaccine of the presentinvention. The seed virus is inoculated in a culture system selectedfrom those which were explained above as usable for the attenuationpassages. The choice may be directed to the same system as, or adifferent system from, the system or systems used for the attenuation ofthe seed virus. In the case where a tissue culture is chosen for theproduction of the vaccine, it is recommended that the virus is allowedto propagate in a culture medium containing none of such substances thatmay act as an antigen when the resulting vaccine is inoculated intohuman body.

From the culture fluid thus obtained, solid matters such as cells, cellfragments or the like are removed, for example, by means of filtrationor centrifugation, and the filtrate or the supernatant fluid can beused, as the product of the present invention, per se or diluted with asuitable diluent such as a physiological salt solution or distilledwater, depending on its virus titer.

While the highly attenuated rubella virus vaccine thus produced is readyfor use, it can be preserved in a frozen form with or without additionof one or more stabilizing agents such as sucrose, lactose, glutimates,

phosphates and the like. Alternatively, it may be lyophilized with orwithout addition of one or more of stabilizing agents such as humanserum albumin, gelatin and the like for its storage, and the lyophilizedproduct is dissolved upon its use with a suitable diluent such asphysiological salts solution or sterile distilled water.

For a satisfactory vaccination, it will be required to inoculate atleast 10 InD (50% interfering dose) of virus titer per person,preferably subcutaneously for all at once. Particularly preferable doseis about 10 to 10 InD A dose higher than this does not give any dangerof undesirable side-effects because of the high safety of the vaccinepresented here, but it will be meaningless to use so high dosage sincethere is not expected any particular increase in the desired vaccinationeffect.

For the subcutaneous inoculation, a dose of the necessary virus titer isto be contained in an aqueous composition of about 0.1 to about 1.0 ml.,desirably 0.25 to 0.5 ml., in volume. Thus, the vaccine of the presentinvention is to be adjusted, at the use, so as to comprise the highlyattenuated rubella virus Strain To336 in a rubella virus titer of atleast 10 InD /ml., desirably about 10 to 10 InD /ml., andphysiologically acceptable carrier therefor.

When the highly attenuated rubella virus vaccine thus produced isinoculated into infants and chlidren, there is subsequently observed aremarkable elevation of the antibody level in serum which will preventthem from infection of rubella virus. When the vaccine is applied toadults, inclusive of women of childbearing age, there is also observedthe increase of the serum antibody in blood, and it is noteworthy thatthere are observed no rubella-like symptoms which are considered to beundesirable side-eifects often occurring in consequence of vaccinationby the hitherto-known rubella virus vaccines. Thus, the novel and highlyattenuated rubella virus vaccine of this invention can be used safelyeven for adults to be effectively immunized against rubella virusinfection.

The safety of the highly attenuated rubella virus vaccine of the presentinvention will be further explained by way of examples of the followingtests. Then, the present invention will be demonstrated in furtherdetails by way of examples. Throughout the specification as well as inthe following tests and examples, abbreviations g., ml. and rpm. meangram(s), milliliter(s) and round(s) per minute, respectively.

TEST 1 (FETAL TOXICITY TEST IN MBBITS) Healthy 8-month-old femaleJapanese white rabbits were mated, and after 8 days from the conception1.0 ml. of a test sample was inoculated into an ear vein of the pregnantrabbits. As the test samples were used the following:

(a) Infected tissue culture fluid of non-attenuated rubella virus StrainTo-336 passaged 3 times with African green monkey kidney (hereinaftermay be referred to as AGMK) cells;

(b) Highly attenuated rubella virus vaccine produced according toExample 1 detailed below;

(c) Infected tissue culture fluid of non-attenuated rubella virus Brownstrain 1 passaged 5 times with AGMK cells (positive control), and

(d) Non-infectious tissue culture fluid of AGMK cells (negativecontrol).

The baby rabbits normally born from the infected mother rabbits wereobserved for 30 days after their birth, and then the surviving babyrabbits were sacrificed for autopsy by macroscopic and histopathologicalobservations. The results of the observations are summarized in Table 1.

In the group (c) where the maternal infection was made with the Brownstrain, only seven baby rabbits survived 1 Brown strain is one of themost popular and typical wild strains of rubella virus in the UnitedStates.

6 among the born fifty-eight, and in four rabbits among the TEST 4(SAFETY TEST) survived seven, there were observed typical malformationssuch as cataracts, defect or iris, corneal opacity, The highlyattenuated rubella virus vaccine produced capillary invasion intocornea, staphyloma and microaccording to Example 1 was subjected totests after the phthalmia, either bilateral or unilateral. In othergroups 5 manner prescribed in Section 73.114 of Public Health ,(a), (b)and (d), 58, 56 and 61 baby rabbits survived, Service Regulations, Title42, Part 73, U.S.A. for safety respectively, all without any appreciablemalformations. tests of poliomelitis vaccine, live, oral, and after theIn the group (0) where the material infection was with manner prescribedin Section 73.73 of the same regulathe Brown strain, most of the 51death cases were tions for sterility tests.

observed within three days subsequent to the birth, and As the resultsof inoculation tests in various animals the death rate among babyrabbits who survived for (i.e. rabbits, adult mice, suckling mice andguinea pigs),

seven days after birth was at the same degree as of other tissue culturetests with various primary cells (i.e. monkey groups. kidney, human,amnion, human kidney and rabbit kidney) TABLE 1 Group Strain 'Io-336To-336 Brown None. Passage history AGMK-3... AGMK--- AGMK5Administration dose 2 2 4 3 3 0 Number of pregnant rabbits.

Number of baby rabbits (N) Number of the baby rabbits died after b th (DDeath rate (DIN) percent Rate of malformations among the survived 1 AGMK means that the passages were with African green monkey kidney cells,and the figure shows the number of the passages. 1 The dose is expressedin terms of common logarithm of 50%-interlenng dose (logiu InDso).

TEST 2 (FETAL TOXICITY TEST IN MICE AND and negative tests ofadventitious agents, it was confirmed RATS) that the tested vaccinesatisfied every requirement of the Healthy 10 to 12-week-old female mice(CF-l) and above'referred regulations rats (Sprague Dawley) wererespectively mated, and after five days from the conception, InD 10 /02ml. of a EXAMPLE 1 tissue culture fluid of Rubella virus, Strain To-336,passaged 3 times with AGMK cells was inoculated intra- Kidneys areremoved aseptlcally from hfialthy Afrlcah venously into the pregnantanimals. On the day before green monkeys The ys are Washed with theexpected day of delivery, i.e. the 19th day of preg- Hanks balanced saltsolut1on and are minced. The

nancy for mice and the 21st day for rats, they were minced tissue issfuspehded in about 50 times hy VolumF cesarotomized and the festuseswere picked out and suba YP PP h Sohlllloh, and 1S jected to macroscopicobservations, including skelton dlgesfed under agltatwn- The resultmgfree cells are stained with Dowsons method (refer to Dowsons, A. B.; 40ileFted by cehtrifugatioh at 1,000 -P- 5 minutes, and s i T h l 1 123 to124 192 and histo. diluted with such amount of a lactalbumin Hankssolupathological findings of eyes and auditory systems. As the tiohis toWhich 5% of inactivated Ca f Serum is Supplenegative control, thenon-infected tissue culture fluid mehted, that the heshltaht C61181151961181011 COIIlaIIlS about was administered. The results of theobservations in com- X cells P mlhlhtef- The shspellslofl 1S incubated Sparison with the negative control are summarized in tlohallly 1h Rouxbottles at Aftel 7 y when the Table 2, from which it is noted that thematernal infeccells have firmly propagated 011 the inside Wall Of thetion with Strain To-336 did not give any abnormalities bottles, the66115 e Washed ee times With TC medium in the fetus s, 199 6 to preparean AGMK cell culture medium.

TABLE 2 Animal species Mouse Rat Group. Control 'Io-336 Control To-336Number of pregnant animals 7 9 12 12 Fetal mortality (percent)(deaths/total implants) 35.0 34.1 9.0 6. 2

Number of living to uses 56 60 152 151 Fetal body weight (g.)(meanistandard error) l.03;i=0. 031 1. 10;l=0.063 3. 1710.063 3.22i0.097

Number of fetuses with skeletal abnormalities 0 0 0 0 Number of fetuseswith visceral defects 0 0 O 0 Histopathological findings of eyes andauditory system Normal Normal Normal Normal TEST 3 ,(INOCULATION TEST INMONKEYS) 60 4 Hanks balanced salt solution consists of 2 The threegroups, each consisting of five healthy NaCl African green monkeys, wererespectively inoculated with C 1 0.4 the highly attenuated rubella virusvaccine which was Magsfifizo 8% produced in Example 1 detailed below andwhich shows glgPOtlHeo 0.03 a virus titer of at least 10 InD /ml.: NaIICOZ 8:3

d-Glucose Phenol red (a) lntrathalamicly 1111., 0-5 ml. each to bothheml' Distilled water (triple distined), an amount to spheres); make thetotal 1 liter. (1)) intraspinally (0.2 ml.); and 5 5 Lagtalbitmllgnlianlks slolutioni isthpreiriiarid lgyldissialvi-nfi g. 0 3.0 8.11111111 y 1'0 yza e D. e an S a 8.110% S3. (c) mtramuscularly solutionto make the total 1,000 ml.

6 TC medium 199 is sterile aqueous solution adjusted to pH The monkeyswere observed for the subsequent 28 7.2 containing amino lacitdds,purine basea p yrngidin bas es,

vitamins, sugars, nuc eo-i es rnlo rganic s s, e. e edays and thenSubjected to mstopathologlcal observa tailed composition is to bereferred to: e.g., Morgan, J. F.

tiOIIS, whereby no abnormalities were foundet a1. Proc. Soc. Exp. Biol.Med. 73, pp. 1 to 8 1950).

A seed virus of Rubella virus, Strain To-33 6, is inoculated into thecells and absorbed into the cells at 37 C. for 90 minutes. Then, thefluids are discarded, and the cells are Washed three times with TCmedium 199. Finally, 40 ml. of TC medium 199 to which 2% of inactivatedcalf serum is supplemented is added to the bottle, and incubation iscarried out at 32 C. for 7 days. The culture fluid is centrifuged at3,000 r.p.m. for 5 minutes to separate a supernatant, which is to beused as the seed virus in the next passageafter dilution by times byvolume of TC medium 199.

In this manner, the passage with the AGMK tissue culture is repeated 24times counted from the stage of isolation of the wild Strain To-336, toobtain a seed virus for the production of a rubella virus vaccine. Theseed virus is inoculated to the cells and incubated in 40 ml. of aserum-free TC medium 199 at 32 C. for 7 days. The culture fluids arecollected and immediately frozen for storage at -70 C. Another 40 ml. ofa fresh serum-free TC medium 199 is added to the remaining culturedcells and the incubation is continued at 32 C. for 24 hours. The culturefluids are again collected and frozen. This procedure is repeated up tothe 10th day of incubation. All of the frozen culture fluids are quicklythawed, pooled and centrifuged at 3,000 r.p.m. for 5 minutes to separatea supernatant, being a highly attenuated rubella virus vaccine. Thesupernatant shows a rubella virus titer of 10 lnD /ml. assayed with thesystem of ECHO- 11 and AGMK.

The product is frozen for storage at -70 C. After 100 day storage, it isthawed and diluted 3 times by volume with physiological salt solution.The diluted vaccine is subcutaneously inoculated to an arm to exhibitthe desirable effect of immunization in human adults as well as inchildren and infants.

EXAMPLE 2 the strain being frequently used for the infectivity assay ofrubella virus.

is inoculated a seed of rubella virus, Strain To-336, passaged 20 timeswith AGMK cells as described in Example 1. The inoculum is incubatedunder the same conditions as stated in Example 1. The passage with theDE cell culture is repeated 4 times, and the culture fluids of the 4thculture is centrifuged to obtain a seed virus for the production of arubella virus vaccine.

The seed virus is inoculated to the DE cells in Roux bottles andprocessed in the same manner as in Example 1 by the use of a serum-freeTC medium 199, to produce a highly attenuated rubella virus vaccine. Thevaccine obtained by the final centrifugation shows a rubella virus titerof 10 InD /ml. assayed with the same system as in Example 1.

'In the same manner as in Example 1, the product is frozen for storage,and thereafter thawed and used for vaccination to give satisfactoryimmunization in human adults as well as in children and infants.

The DE cells used as the tissue culture for the passages and the vaccineproduction are to be negative to the' 'CO'FAL test (refer to e.g.Sarrna, P. S. et al.; Virology, 23, 313 et seq. (1964).

What is claimed is:

1. A highly attenuated Rubella virus vaccine compnsmg:

(a) live rubella virus Strain ATCC-VR-533 subjected to at least 10passages of cultivation in a tissue culture containing living cells of awarm-blooded animal, said cells being capable of supporting said virus,at a temperature of about 28 C. to about 36 C. and ceasing said passageswhen sufficient attenuation is attained.

(b) a physiologically acceptable carrier therefor, its

rubella virus titer being at least 10 InD /milliliter when diluted ininoculatable form.

2. The vaccine according to claim 1 wherein the rubella virus titer isfrom 10 to 10 InD /milliliter.

References Cited Buynak et aL, Journal of the American MedicalAssociation, vol. 204, No. 3, pages 103-108, Apr. 15, 1968.

r RICHARD L. HUFF, Primary Examiner

